The wound repair process of corneal endothelium appears to have two distinct pathways: 1) the regenerative pathway, by which endothelial cells do not replicate but are replaced by migration of exiting cells;and 2) the non-regenerative pathway (or fibrosis), by which transformed endothelial cells not only resume proliferation but alter cell morphology and collagen phenotypes, leading to the production of an abnormal fibrillar extracellular matrix. One clinical example of fibrosis is the formation of a retrocorneal fibrous membrane (RCFM) between Descemet's membrane and the corneal endothelium, the physical presence of which causes loss of vision. The long-term goal of this project is to characterize the mechanism involved in the endothelial mesenchymal transformation (EMT) observed in RCFM. During EMT, three major phenotypes are altered in corneal endothelial cells (CECs): cell proliferation is markedly stimulated;the characteristic contact-inhibited phenotypes are lost;and fibrillar extracellular matrix is produced. In our earlier study, we showed that fibroblast growth factor 2 (FGF-2) is the direct mediator of EMT. During the current funding period, we have determined the molecular mechanisms by which these phenotypes are modulated in response to FGF-2 stimulation. We showed that there are distinct populations of p27Kip1 (p27) that employ differential kinetics of phosphorylation, ubiquitination and degradation, suggesting that CECs under proliferative control exert multiple pathways to remove p27. We also showed that FGF-2 reorganizes actin cytoskeleton using Rho kinases (active Rac and Cdc42, and inactive Rho), thus generating a migratory cell phenotype. Finally, we showed that FGF-2 induces secretion of type I collagen by stabilizing [unreadable]1(I) collagen RNA. In addition, we discovered that interleukin-1B (IL-1B) is also involved in FGF-2-mediated EMT;we showed that IL-1B greatly induces FGF-2 in CECs. We propose that there are distinct pathways during EMT: IL-1 B -and FGF-2-mediated pathways. We further propose that the major role of IL-1 B is to induce FGF-2, while FGF-2 serves as main inducer of EMT. Therefore, it is timely to explore distinct signal transduction triggered by IL-1 B or FGF-2 within the biological contexts of EMT caused by inflammation. Although PI 3-kinase is a major signaling molecule throughout the EMT process, we will further explore other specific signaling pathways. We will test the hypothesis in our in vitro EMT model using experimental protocols (transfection, multiplex protein array, GTP-pulldown assay, co-immunoprecipitation, two-dimensional gel electrophoresis, transcription assay, confocal microscopy, etc). We will investigate;1) the regulatory mechanism of Rac and ERK1/2 on mitogenic pathway in response to FGF-2 stimulation;and 2) the molecular mechanism by which IL-1b induces FGF-2. Such detailed information is crucial for targeting the specific stage and specific pathway before inflammation causes an irreversible EMT.